Title: Nondestructive 3-D imaging of Live Cultures and Tissue Engineered Constructs PI: Todd Doehring Project Abstract Tissue engineering has the potential to revolutionize the clinical treatment of difficult problems such as the repair of diseased aortic valves or articular cartilage. Unfortunately, current tissue engineered constructs still lack the strength, flexibility, and long term durability required for function. In addition, the integration of construct and host tissue is a significant problem. Although much data on the molecular and fibrillar microstructures of fixed and histologically prepared specimens are available, a significant gap remains in our knowledge of the real-time cell morphology changes, and deposition and reorganization of extracellular matrix in live tissue engineered cultures. This is due in part to a lack of tools for non-destructive imaging and real- time analysis of collagen structure in fresh specimens and live tissue constructs. Recently, we have developed a new imaging technique that uses elliptically polarized light microscopy to reveal detailed collagen fiber structures in fresh tissues and cultured specimens, without the need for destructive histological procedures. Preliminary results have provided striking new images of in-situ collagen/cell structure and time-lapse images of live cell-gel preparations (presented here), motivating the new proposed experiments and development of analytical tools for quantifying 3-D fiber-scale tissue structure-function behavior. The goal of this proposal is to apply these novel real-time imaging methods for analyses of a series of cell/scaffold tissue engineered constructs cultured under controlled loading conditions. Broad Impact: Leveraging our imaging and analysis methods and newly available digital imaging systems we intend to provide unprecedented real-time visualizations and analyses of tissue/collagen 3-D structure and live cell-matrix interactions for a wide range of tissues and tissue engineered constructs. Detailed understanding of fiber scale tissue structure and time- dependent changes under controlled loads is critical for researchers and implant designers developing "next generation" tissue engineered repair options for diseased and injured soft tissues. The tools developed here could also have wide impact in understanding the temporal sequence of collagen assembly and cell mediated remodeling/regeneration. Summary of Specific Aims, Hypotheses, and Deliverables: Specific Aim I (Year 1): Adapt and apply our new nondestructive 3-D elliptically polarized light imaging and testing system to analyses of tissue constructs in culture with 'live'time-lapse 3-D imaging of electrospun tissue engineered constructs. Validate our results against existing confocal microscope images. Hypothesis I: 2-D and 3-D cell and matrix morphologies, including volumetric and surface topology parameters, will be statistically similar to static confocal images. Deliverable I: A validated nondestructive real-time system capable of live cell/ECM imaging. Specific Aim II (Year 2): Imaging and analysis of 'live'time-lapse changes in cell-ECM and electrospun tissue engineered constructs under varying controlled tension-flexion experiments. Hypothesis II: 3-D cell and collagen matrix assembly/remodeling morphologies, will vary depending on the scaffold properties and applied tension/flexion loads. Deliverable II: Live, real-time images (video) of cells assembling collagen matrix for a series of tissue engineered scaffolds and during varying loading conditions. Additional validation and analyses using confocal microscopy will also be performed. PUBLIC HEALTH RELEVANCE: Project Narrative Tissue engineering has the potential to revolutionize the clinical treatment of difficult problems such as the repair of diseased aortic valves or articular cartilage. Unfortunately, current constructs do not have adequate biomechanical properties or fiber structure. Monitoring tissue cell-ECM structure during culture is challenging. There is a lack of tools for non-destructive imaging and real-time analysis of collagen structure in fresh specimens and live tissue constructs. Recently, we have developed a novel imaging technique that uses elliptically polarized light microscopy to reveal detailed collagen fiber structures in fresh tissues and cultured specimens, without the need for destructive histological procedures. The goal of this proposal is to apply these novel real-time imaging methods for analyses of a series of cell/scaffold tissue engineered constructs cultured under controlled loading conditions. High resolution, long term time-lapse images and analysis of collagen assembly and fibrogenesis in tissue engineered constructs could be highly valuable for development of new methods for improving tissue engineered construct biomechanical and biochemical properties, as well as an improved understanding of cell-ECM interactions.